Latitudinal variation of the polymorphic sn-glycerol-3-phosphate (α-Gpdh) locus in Drosophila melanogaster has been characterized on several continents; however, apparent clinal patterns are potentially confounded by linkage with an inversion, close associations with other genetic markers that vary clinally, and a tandem α-Gpdh pseudogene. Here we compare clinal patterns in α-Gpdh with those of other linked markers by testing field flies from eastern Australian locations collected in two separate years. The α-Gpdh variation exhibited a consistent non-linear cline reflecting an increase in the α-GpdhF allele at extreme latitudes. This pattern was not influenced by the In(2L)t inversion wherein this locus is located, nor was it influenced by the presence of the α-Gpdh pseudogene, whose presence was ubiquitous and highly variable among populations. The α-Gpdh pattern was also independent of a cline in allozyme frequencies at the alcohol dehydrogenase (Adh) locus, and two length polymorphisms in the Adh gene. These results suggest clinal selection at the α-Gpdh locus that is partially or wholly unrelated to linear climatic gradients along the eastern coast of Australia.